1. Field of the Invention
The present invention relates to stable liposomal vectors, in pulverulent form, for active principles, and more particularly for active principles which are sensitive to digestive and/or plasmatic degradation, such as proteins, and to their use as medicinal products.
2. Description of the Background
Many vectors have been proposed to protect such fragile active principles; among these, mention should be made of liposomes, which have been considered as a vector of choice.
The first studies on the oral administration of liposomes were not conclusive (Deshmukh D. S. et al., Life Sciences, 1981, 28, 239-242). The results obtained showed that liposomes with the formulation: diether-phosphatidylcholine (indigestible PC analogues)/cholesterol-7:1 allowed gastrointestinal protection of the encapsulated peptide, but did not allow its passage across the intestinal barrier.
Several reasons may be put forward to explain this absence of passage: excessively large and non-calibrated size of the liposomes, low stability of the structure or leakage of the encapsulated compound into the extra-liposomal medium.
Recently, the research team of Robert Greenwood (Drug Dev. and Ind. Pharm., 1993, 19, 11, 1303-1315) at the Campbell University, U.S.A., has succeeded in showing that the duodenal intubation of liposomes vectorizing insulin brought about a higher hypoglycaemiant effect than that obtained after a duodenal intubation of a solution of free insulin.
Many tests have been carried out to obtain liposomes with good capacity to transport active principles, in particular as regards the action on the percentage of uptake of the active principle, the stability of the liposomes and the bioavailability of the active principle. Mention may be made, for example, as a guide, of:
S. B. Kulkarni et al. (J. Microencapsulation, 1995, 12, 3, 229-246) who point out the factors involved in the microencapsulation of medicinal products in liposomes: size of the liposome, type of liposome, surface charge of the liposome, rigidity of the bilayer, addition of encapsulation adjuvants. It emerges from this evaluation that MLVs (multilamellar vesicles) containing several bilayers and with a diameter of between 100 nm and 20 mm are desirable for the encapsulation of hydrophobic medicinal products interacting with the bilayers, whereas LUVs (large unilamellar vesicles) containing a single bilayer and with a size of between 100 and 1000 nm are considered as being the most suitable for the encapsulation of hydrophilic medicinal products.
I. De Miguel et al., (Biochimica et Biophysica Acta, 1995, 1237, 49-48 [sic]), who propose nanoparticles composed of an internal core formed from crosslinked polysaccharides grafted on their exterior with fatty acids and surrounded by a layer of phospholipids;
P. S. Uster et al., (FEBS Letters, 1996, 386, 243-246) who propose the insertion of phospholipids modified with a poly(ethylene glycol) in preformed liposomes to improve the bioavailability.
Series of experiments relating to the oral administration of peptides have been carried out and use either different liposomal methods of encapsulation, or modification of the lipidic active principle by grafting lipophilic functions. In all cases, the aim is to convert the lipidic active principle into a xe2x80x9cprodrugxe2x80x9d; this prodrug has the property of withstanding gastrointestinal transit, i.e. resistance to gastric pH, to physiological detergents (bile salts), to proteases (intestinal exopeptidases and endopeptidases) and to metabolization by the intestinal flora. For example, the bridging in position 2 of a 1,3-diglyceride onto a pentapeptide made it possible to impart these qualities to the drug thus modified.
However, these various liposomes of the prior art do not make it possible to obtain both good stability, an acceptable active-principle encapsulation yield and a significantly improved oral bioavailability of the said active principle, without modifying the active principle, which thus conserves all of its functions and properties. The term xe2x80x9cbioavailabilityxe2x80x9d means the fraction of the dose which reaches the systemic circulation in pharmacologically active form and the rate at which it does so.
J. C. Hauton has described liposomes with a gelatinized internal core (lipogelosomes(copyright)) which are in suspension in aqueous medium containing gelatinizing substances. He has, in particular, developed a process for manufacturing such liposomes (European patent 0 393 049), which differ from conventional liposomes in that the encapsulated aqueous phase is in semi-solid gel form rather than in liquid form, and this prevents the liposomes from fusing during collisions. Such lipogelosomes(copyright) are produced entirely from natural substances, thereby minimizing the risk of intolerance. In particular, in European patent 0 393 049, these lipogelosomes(copyright) consist of one bilayer interfacial phase, in the case of the unilamellar lipogelosomes, or of a plurality of bilayer interfacial phases, which are superimposed concentrically, in the case of the multilamellar lipogelosomes(copyright), and of a gelatinized encapsulated internal aqueous polar phase in which the gelatinized substance, which may or may not be polymerizable, is selected from polysaccharides, polypeptides or polyacrylamides; for example, the non-polymerizable gelatinizable substance is selected from gelatin, agarose or carrageenans, and the polymerizable gelatinizable substance is selected from polyacrylamide gels. These lipogelosomes(copyright) possess a stability which is significantly increased as compared with the liposomes of the prior art, particularly because of the absence of interparticulate fusion during collisions.
However, they suffer from the drawback of being in the form of a dispersion of liposomes in liquid phase, which is not suitable for preparing solid formulations which are easy to store and to administer.
Consequently, the Applicant set itself the objective of providing a novel vector which effectively makes it possible to obtain both a sufficient encapsulation yield and significantly improved oral bioavailability of the said active principle, compared with the liposomes of the prior art, while at the same time displaying great stability both on storage and in vivo. Said vectors are suited to oral administration; the aqueous solution is also suitable for other routes of administration: transdermal, pulmonary, nasal, genital, intravenous, subcutaneous or ocular, for example, depending on the excipient selected.
The said vectors are characterized in that they consist of:
a pulverulent composition which consists essentially of unilamellar liposomes comprising an external lipid phase which consists of class 4 lipids (phospholipids), optionally combined with class 2 substances (long-chain triglycerides, cholesterol esters), class 3 substances (cholesterol, nonionized long-chain fatty acids) and/or class 5 substances (bile salts, fusidic acid derivatives) and an internal aqueous core forming a temperature-reversible aqueous gel which radiates out up to the external lipid phase, which internal aqueous core essentially consists of a mixture M of at least two different non-polymerizable gelatinizing agents G1 and G2 whose gel-sol phase transition point is higher than or equal to 37xc2x0 C., with G1 being a gelatinizing agent which is selected from gelatins and carrageenans, such as kappa-carrageenans, and G2 being selected from carrageenans whose properties are different from the carrageenans selected for G1, such as iota-carrageenans, and celluloses, such as hydroxypropylmethylcellulose, which liposomes have a diameter of between 20 nm and 1 mm, preferably of between 20 nm and 500 nm and being in the form of particulate units with an average diameter of between 10 mm and 1000 mm, formed from one or more of the said liposomes, surrounded by a matrix selected from the group consisting of a dehydrated temperature-reversible aqueous gel which is identical to the aqueous gel of the said internal core, dextrins or a mixture thereof, such that it comprises, on average, 1016 to 1018 liposomes/g of powder, and
at least one active principle included, depending on the case, either in the gelatinized internal core or in the external lipid phase of the said composition.
Surprisingly, such vectors make it possible to overcome the drawbacks associated with conventional liposomes. Specifically, they make it possible:
to increase the stability of the liposomes, on account of the absence of interparticulate fusion during collisions;
to increase the bioavailability of the active principle (protection in the gastrointestinal tract and passage across the intestinal barrier); in particular, in rats, the passage time of the vectors according to the invention (LGS) across the intestinal barrier from the moment of their oral administration can be between 2 and 4 hours: i.e. 1 hour of gastric emptying and 1 to 3 hours of passage from the intestinal lumen into the systemic circulation; thus, an active principle whose cellular internalization capacity is low or non-existent can be incorporated effectively into a differentiated intestinal epithelial cell, when it is encapsulated in a vector (LGS) according to the invention, without modifying the activity or composition of the active principle;
to reduce the toxicity of the the encapsulated active principles; and
to result in fewer leakages of the encapsulated products, on account of the lower molecular mobility in the gelatinized encapsulated aqueous phase.
Unexpectedly, by selecting the gelatinizing agents, it is possible to obtain liposomes (SUVs or small unilamellar vesicles), which are suitable for use in a dry form (powder) and which have particularly advantageous properties as vectors for active principles; in specific terms, surprisingly, the oral bioavailability of the said active principlesxe2x80x94preferably of active principles which are sensitive to digestive degradation, poorly absorbed or highly toxicxe2x80x94is significantly increased when they are encapsulated or combined with the vector according to the present invention.
In addition, such vectors in pulverulent form conserve all the integrity of the liposomes they contain, which remain stable over time, both in pulverulent form and when they are suspended, on account of the maintenance of the integrity of the constituent lipids (no degradation product) and the maintenance of the integrity of the characteristics of the gelatinizing agents, in particular of the mixture G1 and G2 (viscosity, gel strength and breaking force, molecular masses).
The advantage of using lipogelosomes(copyright) (LGS) in this context is that of benefiting from a stabilized liposomal form (J C Hauton et al., Eur. J. Surg., 1994, suppl. 574, 117-119) for the purpose of the oral administration of active principles. The method for manufacturing LGSs makes it possible to obtain, on average, degrees of encapsulation of the gelatinized hydrophilic phases of close to 10%. This percentage varies, in particular as a function of the molecular weight of the active principle, and is calculated according to the ratio: amount of active principle encapsulated/amount of active principle used. For example, at least 5% encapsulation is observed for a 500 Da molecule and at least 50% encapsulation is observed for a molecule of at least 20 kDa. As regards peptides, for example, 10 to 50% encapsulation is observed, whereas, in general, for active principles as a whole, the percentage of encapsulation ranges from 5 to 80%, depending on the case.
The gelatinizing agents G1 and G2 differ in particular as regards the viscosity, molecular mass and gel-sol transition point (i.e. the melting point). For the gelatinizing agents C1, this temperature is less than or equal to 45xc2x0 C., whereas it is greater than or equal to 45xc2x0 C. for the gelatinizing agents G2.
The mixture M of at least two gelatinizing agents G1 and G2 as defined above has texturometric characteristics (gel strength and breaking force) which are particularly advantageous from the point of view of the stability of the liposomes obtained and the bio-availability of the encapsulated active principle. Thus, the mixture M of at least two gelatinizing agents G1 and G2 preferably has, at 5xc2x0 C., relaxation characteristics of between 70 and 100%, preferably 81-89% and a breaking force of between 1000 and 1600 g, preferably 1109-1503 g.
According to another advantageous embodiment of the said composition, the said internal aqueous core of the liposomes also comprises at least one stabilizer of glycosidic nature, and/or at least one agent for regulating the osmolarity of the medium and/or at least one surfactant, such as a bile salt and/or a nonionic surfactant.
Advantageously, the said vectors comprise, as % (m/m): 25 to 75% of class 4 lipids, 5 to 45% of gelatinizing agents, 0 to 70% of stabilizer of glycosidic nature, 0 to 15% of agent for regulating the osmolarity of the medium, 0 to 20% of surfactants and 0 to 15% of dextrins, preferably 8 to 12%; this formulation does not include the active principles.
According to another advantageous embodiment of the said pulverulent composition according to the invention, the said aqueous internal core comprises 70 to 95% of gelatinizing agent G1 and 5 to 30% of gelatinizing agent G2.
According to another advantageous embodiment of the said pulverulent composition according to the invention, the stabilizer of glycosidic nature is sucrose, trehalose or any other protective agent.
The subject of the present invention is also a process for preparing the pulverulent vectors according to the invention, in which the external matrix of the particulate units comprises a fraction of temperature-reversible aqueous gel, characterized in that it comprises the following steps:
(1) preparation of a dispersion of liposomes with a gelatinized internal core (lipogelosomes(copyright)) in aqueous phase by (a) preparing a solution of at least one suitable gelatinizing agent, in particular a mixture M of gelatinizing agents G1 and G2, by dissolving the said gelatinizing agents, with slow stirring, at a temperature above the gel-sol phase transition temperature of the said gelatinizing agents, in an aqueous solution whose pH is compatible with the active principle to be encapsulated, (b) incorporating the active principle into the solution obtained in (a), (c) incorporating the lipids into the solution obtained in (b), with slow stirring of the mixture, for a period of less than 5 hours, preferably under vacuum, and formation of an emulsion, and (d) obtaining the said dispersion of liposomes with a gelatinized internal core (lipogelosomes(copyright)) in an aqueous phase containing the said gelatinizing agents, by rapid stirring of the emulsion obtained in (c), preferably under vacuum, and
(2) production of the pulverulent product by suitable drying of the dispersion obtained.
According to one advantageous embodiment of the said process, the drying is carried out by atomization, coacervation, thin layer or granulation.
Another subject of the present invention is a process for preparing the pulverulent vectors according to the invention, in which the external matrix of the particulate units comprises a fraction of temperature-reversible aqueous gel and/or a dextrin, characterized in that it comprises the following steps:
(1) preparation of a dispersion of liposomes with a gelatinized internal core (lipogelosomes(copyright)) in aqueous phase by (a) preparing a solution of at least one suitable gelatinizing agent, in particular a mixture M of gelatinizing agents G1 and G2, by dissolving the said gelatinizing agents, with gentle stirring, at a temperature above the gel-sol phase transition temperature of the said gelatinizing agents, in an aqueous solution whose pH is compatible with the active principle to be encapsulated, (b) incorporating the active principle into the solution obtained in (a), (c) incorporating the lipids into the solution obtained in (b), with slow stirring of the mixture, for a period of less than 5 hours, preferably under vacuum, and formation of an emulsion, and (d) obtaining the said dispersion of liposomes with a gelatinized internal core (lipogelosomes(copyright)) in an aqueous external phase containing the said gelatinizing agents, by rapid stirring of the emulsion obtained in (c), preferably under vacuum, and
(2) at least partial removal of the aqueous liquid phase containing the said gelatinizing agents, in which the liposomes are dispersed,
(3) addition of at least one suitable dextrin, and
(4) production of the pulverulent product by drying by atomization of the product obtained in (3).
According to one advantageous embodiment of the said process, step (2) of at least partially removing the aqueous liquid phase containing the said gelatinizing agents is carried out by dilution and/or filtration.
In accordance with the preparation processes according to the invention, the aqueous solution in step (a) also comprises an agent for regulating the osmolarity of the medium (for example 0.9% NaCl) and/or a stabilizer of glycosidic nature and/or a surfactant, preferably class 5 substances (bile salts).
As a variant, the active principle is added to the external lipid phase before it is incorporated into the mixture obtained in (a).
For example, calcitonin is incorporated at pH 5, AZT is incorporated at pH 7.5 and doxorubicin is incorporated at pH 3.
Surprisingly, such processes make it possible to obtain a vector in pulverulent form based on stable liposomes with a gelatinized internal core (lipogelosomes(copyright)) in the course of a single step comprising a phase of maturation (in the sense of ripening) of the constituents in aqueous phase, at slow speed, followed by a phase of dispersion (formation of the lipogelosomes(copyright)) at high speed, comprise a step during which a stable dispersion of lipogelosomes(copyright) in liquid phase, of homogeneous morphology, is obtained, which can be subjected to the drying step; such a dispersion of liposomes with a gelatinized internal core effectively has the following morphology:
vesicular structure with a diameter of between 20 nm and 500 nm, preferably between 20 and 80 nm,
negative staining microscopic observations, cryofracture, cryotransmission and atomic force: vesicles or assemblies of vesicles with the characteristic appearance of phospholipid bilayers; negative staining makes it possible to observe the more or less pronounced presence of a mixture M of gelatinizing agents enveloping the external phospholipid layer, and
polydispersity of the liposomes with a gelatinized internal phase of between 10 and 55%, preferably between 10 and 30%.
Such a process has the advantage of being reproducible and fully adaptable to the industrial scale.
It also has the advantage of being less cumbersome to implement than the processes of the prior art in which a step of sonication, extrusion or removal of detergents is necessary, as described in patent 0 393 049.
According to one advantageous embodiment of the said processes, the step (c) is preferably carried out at a shear rate of less than 200 sxe2x88x921; in general, the shear rate is given by the following ratio: speed of the stirring unit/space between the internal wall of the reactor and the distal end of the stirring blade (also known as the xe2x80x9cair gapxe2x80x9d).
Another subject of the present invention is a pharmaceutical composition, characterized in that it comprises a pulverulent liposomal active-principle vector as defined above and at least one pharmaceutically acceptable vehicle.
According to one advantageous embodiment of the said composition, it is in solid form (gel capsule, tablet or powder to be dissolved in water).
According to another embodiment of the said composition, it also comprises a cAMP activator.